Carotenoids are synthesized de novo in bacteria, algae, fungi and plants. Carotenoids are a class of natural fat-soluble pigments found principally in plants, algae, and photosynthetic bacteria, where they play a critical role in the photosynthetic process. They also occur in some non-photosynthetic bacteria, yeasts, and molds, where they may carry out a protective function against damage by light and oxygen. Although animals appear to be incapable of synthesizing carotenoids, many animals incorporate carotenoids from their diet. Within animals, carotenoids provide bright coloration, serve as antioxidants, and can be a source for vitamin A activity (Ong and Tee 1992; Britton et al. 1995).
Carotenoids are responsible for many of the red, orange, and yellow hues of plant leaves, fruits, and flowers, as well as the colors of some birds, insects, fish, and crustaceans. Some familiar examples of carotenoid coloration are the oranges of carrots and citrus fruits, the reds of peppers and tomatoes, and the pinks of flamingoes and salmon (Pfander 1992). Some 600 different carotenoids are known to occur naturally (Ong and Tee 1992), and new carotenoids continue to be identified (Mercadante 1999).
Carotenoids are defined by their chemical structure. The majority carotenoids are derived from a 40-carbon polyene chain, which could be considered the backbone of the molecule. This chain may be terminated by cyclic end-groups (rings) and may be complemented with oxygen-containing functional groups. The hydrocarbon carotenoids are known as carotenes, while oxygenated derivatives of these hydrocarbons are known as xanthophylls. Beta-carotene, the principal carotenoid in carrots, is a familiar carotene, while Lutein, the major yellow pigment of marigold petals, is a common xanthophyll.
The structure of a carotenoid ultimately determines what potential biological function(s) that pigment may have. The distinctive pattern of alternating single and double bonds in the polyene backbone of carotenoids is what allows them to absorb excess energy from other molecules, while the nature of the specific end groups on carotenoids may influence their polarity.
The economic importance of carotene compounds in general and in particular ß-carotene and lutein has increased steadily in recent times. Industry has attempted to respond to the stimulated demand on the one hand by synthethic production of carotenoids and on the other hand by extracting and subsequently crystallizing carotenoids from natural sources. The consumers in accordance with their present critical attitude towards synthetic products have a clear preference for natural ß-carotene and lutein.
ß-Carotene for example is a vitamin A precursor and thus an important constituent in food, feed and cosmetic applications. It further serves as a pigmenting substance in many fields, such as, for example, in the beverages industry. Pure carotenoid crystals derived from Marigold flowers, comprising predominantly of Xanthophylls such as Lutein, Zeaxanthin and Cryptoxanthin and low levels of beta-carotene have been proven scientifically to reduce the risk of age related macular degeneration (Reference: Moeller S M, Jacques P F, Blumberg J B “The potential role of dietary Xanthophylls in cataract and age related macular degeneration,” Journal of the American College of Nutrition, 2000; 19: 522S-527S), control over LDL cholesterol (Reference: Chopra M., Thurnham D I, “Effect of Lutein on oxidation of low density lipoproteins (LDL) in vitro”, Proceedings of the Nutrition Society, 1994; 53: 1993, #18A.), prevention of Coronary heart diseases (Reference: Howard A N, Williams N R, Palmer C R, Cambou J P, Evans A E, Foote J W, et al., “Do hydroxy-carotenoids prevent coronary heart disease?” A comparison between Belfast and Toulouse, “International Journal of Vitamin and Nutrition Research, 1996; 66: 113-118) and free radicals scavenging and immunity enhancing (Reference: Chew B P, Wong M W, Wong T S, “Effects of Lutein from Marigold extract on immunity and growth of mammary tumors in mice,” Anticancer Research, 1996; 16: 3689-3694). Lutein, (beta-e-carotene-3-3′-diol) and Zeaxanthin (beta-beta-carotene-3-3′-diol) belong to Xanthophylls group in the carotenoids family with highly reactive hydroxyl groups which cannot be synthesized by humans and animals.
Whereas until recently only the “classical” natural ß-carotene sources such as e.g. carrots or algae were available for commercial isolation processes, innovative biotechnological approaches have nowadays exploited a considerably more suitable profound source using fermentative methods. The fermentation of particular filamentous fungi has enabled a concentration of up to more than 5% by weight ß-carotene to be achieved in the dried fermentation biomass; the concentration of ß-carotene is therefore about ten higher than in the traditional natural sources.
In general, the induction of ß-carotene crystallization by adding solvents invariably leads to high yields, however, it is necessary to add large amounts of solvent as described for n-propanol for example in the U.S. Pat. No. 1,988,031 in order to obtain a satisfying yield. When extracting from natural materials using organic solvents such as e.g. petroleum ether (cf. the U.S. Pat. Nos. 1,967,121 and 1,998,031) there is generally the problem that, due to the low solubility of ß-carotene, and in particular in the case of high ß-carotene concentrations, an extremely large amount of solvent must be selected which, however, in turn considerably and negatively effects the space-time yield.
Recently, several processes have been described in which ß-carotene is extracted from natural materials using supercritical carbon dioxide at very high process pressures (U.S. Pat. No. 4,400,398). Despite the good extraction results a general disadvantage of this gas extraction process is the complicated technical implementation of the required high pressure, which is generally more cost-intensive than processes which operate under normal pressure.
U.S. Pat. No. 5,382,714 reports that saponified marigold oleoresin from Kemin Industries (Des Moines, Iowa) containing free lutein is the preferred starting material for the isolation of pure lutein. The saponification step involves high percentage of propylene glycol and the saponification time is done for a minimum period of three hours subjecting the product to heat for prolonged period, which increases the process time too.
Within the framework of the unification of the food law legislation within the EU, a draft of a guideline to lay down specific purity criteria for dyes, which can be used in foods, was submitted to the commission in January 1995. In this document the solvents acetone, methyl-ethylketone, methanol, ethanol, propan-2-ol, hexane, dichloromethane and carbon dioxide are proposed for extracting natural carotenes. However, with the exception of dichloromethane, these solvents are less suitable for economic extraction from natural materials in which ß-carotene occurs in high concentrations due to their low dissolving capacity for ß-carotene. On the other hand a forward-looking food industry should refrain from using dichloromethane for ecological and consumer-related reasons.
The object of the present invention is therefore to provide novel methods for isolating carotenes, in particular ß-carotene and/or lutein, from new natural sources and in particular from solid natural materials which circumvents the disadvantages of the known processes, in particular for the use as animal feed ingredients.